Biased G protein-coupled receptor (GPCR) agonists are orthosteric ligands that possess pathway-selective efficacy, activating or inhibiting only a subset of the signaling repertoire of their cognate receptors. Unlike conventional agonists that work by changing the quantity of receptor efficacy, biased agonists possess the ability to qualitatively change signaling, suggesting it may be possible to exploit ligand bias to develop drugs that maximize clinical effectiveness while minimizing side effects. Our finding that an arrestin pathway-selective biased agonist for the type 1 parathyroid hormone receptor (PTH1R) promotes anabolic bone formation in vivo without stimulating bone resorption or producing hypercalcemia offers proof of principal that biased agonists can be used to elicit biological responses that cannot be achieved with conventional agonist or antagonist drugs. Using bioinformatic metabolic pathways and gene cluster analysis, we found that the arrestin-selective PTH1R agonist produces a unique genomic 'footprint' at the tissue level, i.e. that its mechanism of action is distinct from that of a conventional agonist and could not have been predicted on the basis of existing knowledge of classical GPCR signaling. This key observation raises fundamental questions about the nature of biased agonism, which we propose to address in this application. We will employ in vitro ligand efficacy profiling, microarray-based functional genomic analyses, and cell-based assays of biological response, to address three Specific Aims. Aim 1 will employ primary calvarial osteoblasts from wild type and -arrestin2-/- mice and PTH1R ligands to determine the functional characteristics of conventional, G protein pathway-selective, and arrestin pathway-selective agonists in a common cell type. We hypothesize that both G protein-selective and arrestin-selective biased agonists will produce genomic footprints that are qualitatively different from a conventional agonist. Aim 2 will employ primary renal tubular epithelial cells and arrestin pathway- selective biased ligands for the angiotensin AT1AR, vasopressin V2R, and PTH1R to determine the effects of activating arrestin signaling pathways via different GPCRs in a common cell background. We hypothesize that the response to different arrestin-selective agonists will overlap, whereas the responses to conventional agonists will diverge due to the concomitant activation of different G proteins. Aim 3 will employ primary osteoblasts, renal tubular epithelial cells and cardiomyocytes, and arrestin pathway-selective biased ligands for the AT1AR and PTH1R to determine the effects of activating arrestin signaling pathways via a common GPCR in different cell types. We hypothesize that activation of arrestin signaling will produce similar effects in different cell backgrounds, reflecive of a limited arrestin-dependent signaling repertoire. This work will establish the range of effects that can be achieved using arrestin-selective agonists, and determine the extent to which arrestin-dependent effects are conserved across different GPCRs and target tissues. This information will be critical to efforts to develop novel therapeutics that exploit ligand bias.